Project summary Breast cancer remains one of the leading causes of death in women and most patients die from the tumor metastasis. Our long-term goal is to understand the molecular mechanisms of metastasis so that novel therapeutic strategy can be designed. EMT (epithelial to mesenchymal transition) is a determining step for a cancer cell to progress from a non-invasive to invasive state. Disruption of intercellular adhesion via loss of E- cadherin, the defining event for EMT, is often correlated with gain of the matrix metalloproteinases (MMP) that catalyze proteolytic matrix remodeling important for tumor cell invasion. Such a correlation has been found in invasive breast cancer. However how EMT is regulated during and contributes to breast cancer metastasis is largely unknown. We previously identified Kr[unreadable]ppel-like transcription factor 8 (KLF8) as a downstream effector of FAK that plays a crucial role in breast cancer metastasis. Recently, we found that KLF8 is important for oncogenic transformation and overexpressed in invasive human breast cancer, suggesting an uninvestigated role for KLF8 in breast cancer metastasis. Our preliminary studies, using inducible and constitutive expression of KLF8 in MCF-10A cells, suggest that KLF8 is a potent inducer of EMT and invasion by repressing E- cadherin and activating MMP9, MMP2 and MT1-MMP. We also show that KLF8 plays a key role in the loss of E-cadherin and gain of the MMPs in invasive human breast cancer cells and that aberrant KLF8 elevation is tightly correlated with the loss of E-cadherin in patient specimens. Finally, our pilot studies suggest a role for KLF8 in supporting MDA-MB-231 lung metastasis. These results strongly support our hypothesis that KLF8 promotes breast cancer metastasis by initiating EMT and invasion through repression of E-cadherin and activation of the MMPs. We propose three specific aims to test this hypothesis using in semi-in vivo 3D acinus culture and in vivo mouse models and our MCF-10A cells expressing inducible KLF8 and MDA-MB-231 cells expressing inducible KLF8 sh RNA. In Aim 1, we will determine the role of E-cadherin and the MMPs in KLF8- induced EMT and invasion by genetically modifying these proteins. In Aim 2, we will identify the molecular mechanisms by which E-cadherin, the MMPs and intergrin-FAK signaling interplay to regulate KLF8-induced EMT and invasion using genetic, biochemical or pharmacological approaches. In Aim 3, we will determine the role of KLF8 in tumor metastasis using a mouse model of human breast cancer and bioluminescence imaging. We will also evaluate role for other EMT regulators such as Snails, ZEBs and Twist in the regulation of metastasis by KLF8. Completion of this project will shed new lights on mechanisms responsible for breast cancer metastasis which may leads to new therapeutic intervention.